The effect of sampling procedures and day-to-day variations in metabolomics studies of biofluids

Metabolomics analysis of biofluids is a feasible tool for disease characterization and monitoring due to its minimally invasive nature. To reduce unwanted variation in biobanks and clinical studies, it is important to determine the effect of external factors on metabolic profiles of biofluids. In this study we examined the effect of sample collection and sample processing procedures on NMR measured serum lipoproteins and small-molecule metabolites in serum and urine, using a cohort of men diagnosed with either prostate cancer or benign prostatic hyperplasia. We determined day-to-day reliability of metabolites by systematic sample collection at two different days, in both fasting and non-fasting conditions. Study participants received prostate massage the first day to assess the differences between urine with and without prostate secretions. Further, metabolic differences between first-void and mid-stream urine samples, and the effect of centrifugation of urine samples before storage were assessed. Our results show that day-to-day reliability is highly variable between metabolites in both serum and urine, while lipoprotein subfractions possess high reliability. Further, fasting status clearly influenced the metabolite concentrations, demonstrating the importance of keeping this condition constant within a study cohort. Day-to-day reliabilities were however comparable in fasting and non-fasting samples. Urine sampling procedures such as sampling of first-void or mid-stream urine, and centrifugation or not before sample storage, were shown to only have minimal effect on the overall metabolic profile, and is thus unlikely to constitute a confounder in clinical studies utilizing NMR derived metabolomics.